BRAF (“v-raf murine sarcoma viral oncogene homolog B1”) belongs to a family of serine-threonine protein kinases and plays a critical role in the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway. Mutations in BRAF are associated with multiple cancers, including colorectal cancer (CRC), thyroid cancer, non-small cell lung cancer (NSCLC), melanomas, and adenocarcinomas (Davies H, et al. Nature 2002; 417:949-54; Rajagopalan H, et al., Nature 2002; 418:934.). BRAF is mutated in over 50% of melanomas, approximately 45% of thyroid cancers, and approximately 10% of colorectal cancers. BRAF mutations are also associated with LEOPARD syndrome, a developmental disorder with an increased incidence of multiple granular cell tumors.
The most prevalent BRAF mutation is a single amino acid substitution at position 600 of the BRAF protein, which is a valine in the wild-type molecule (V600). The most common BRAF V600 mutation is a glutamic acid substitution (V600E), comprising approximately 80-90% of all V600 mutations. The V600E mutation is caused by a T to A transversion in exon 15 of the BRAF gene, at nucleotide 1799 of the coding sequence given by NCBI Accession number NM_004333.4 (1799 T>A). The V600E mutation has been observed in over half of all microsatellite-unstable carcinomas and in a subset of stable colon tumors (Wang L, et al., Cancer Res 2003; 63:5209-12).
The V600E BRAF mutation is also frequently found in tumors of the nervous system, thyroid, skin, gastrointestinal tract, large intestine, biliary tract, ovary, eye, prostate, central nervous system, liver, small intestine, breast, pancreas, soft tissue, digestive tract, adrenal gland, autonomic ganglia, hematopoietic and lymphoid tissue, lung, esophagus, pituitary, and stomach.
The second most common BRAF V600 mutation is a lysine substitution (V600K), which is caused by a GT to AA transversion at nucleotides 1798-1799 of the BRAF coding sequence given by NCBI Accession number NM_004333.4 (1798-1799 GT>AA). Other V600 mutations include, but are not limited to, glutamine (V600R), aspartic acid (V600D), alanine (V600A), and methionine (V600M) substitutions.
Patients harboring BRAF V600K mutations have been shown to respond better to the BRAF inhibitor vemurafenib than to dacarbazine chemotherapy. Likewise, patients harboring BRAF V600E or V600K mutations have been shown to respond better to the mitogen-activated protein kinases kinase (MEK) inhibitor trametinib than to dacarbazine or paclitaxel chemotherapy.
Studies have shown that BRAF mutations are associated with enhanced and selective sensitivity to MEK inhibition compared to wild-type cells and cells harboring a RAS mutation. The effect is observed in BRAF mutant cells regardless of tissue lineage, and correlates with both down regulation of cyclin D1 expression and the induction of G1 arrest. Pharmacological MEK inhibition completely abrogates tumor growth in BRAF mutant xenografts (Solit, D. et al., Nature 2006; 439:358-362). These data suggest a dependency on MEK activity in BRAF mutant tumors, and point to a potential therapeutic strategy for this genetically defined tumor subtype.
A recent study reported the presence of the BRAF gain-of-function mutation V600R in synovial fibroblasts from rheumatoid arthritis patients, and demonstrated that BRAF-specific siRNA inhibits proliferation of the mutant fibroblasts. These findings suggest a mechanism for the fibroblast transformation associated with this condition, as well as a new target for therapeutic intervention (Weisbart, R. H. et al., 2010; 285(45): 34299-34303).
Roche has developed Cobas® 4800 BRAF V600 Mutation Test, which is a companion diagnostic for determining patient eligibility for Zelboraf (or vemurafenib) treatment. Zelboraf was approved by the FDA for the treatment of BRAF V600E mutation-positive inoperable or metastatic melanoma. The Cobas® test detects not only the V600E mutation, but also other V600 mutations such as V600K and V600D.
Several other methods known in the art may be used to detect BRAF V600 mutations, such as Sanger sequencing, which is currently used in clinical settings. A recent study comparing the sensitivity of the Cobas® 4800 BRAF V600 Mutation Test with Sanger sequencing found that the Cobas® test was less sensitive and less specific in detecting V600 mutations than Sanger sequencing (Qu et al., Journal of Molecular Diagnostics 2013; 15(6): 790-795). These findings indicate that the Cobas® assay can miss many V600 mutations in clinical specimens, and that additional assays are required to confirm negative Cobas® results.
Few advances have been made in protocols designed to evaluate patients with BRAF mutations such as V600E and V600K. Identifying specific BRAF mutations will help physicians determine the best treatment strategies and maximize the number of patients who can benefit from treatment with BRAF inhibitors, such as Zelboraf.